Abstract Chromatin is of fundamental importance to basic biological processes that rely on access to the genome. Given that there is such diversity in chromatin structure, a central question is: how is chromatin structure established and maintained within a cell? This question is perhaps most significant during S-phase when chromatin is disassembled and reassembled on the new daughter genomes. We have developed new tools with which we have investigated how DNA replication proceeds across a genome and how chromatin is established on nascent DNA. Our most recent published work has shown that nucleosomes are rapidly organized on newly replicated DNA, but the mechanics of how this happens remain unclear. In this proposal we will generate data to provide a basic framework for understanding how chromatin structures are established in S-phase in budding yeast. Faithful duplication of chromatin during S-phase relies on two pools of histones: those from the parental DNA and those synthesized during S-phase. How the ?old? and ?new? histones are handled by the replisome and deposited into chromatin is not well understood. Recent data has suggested that old histones may be asymmetrically segregated to one daughter cell! ? raising the possibility that old (or new) histones carry important information that help define cell identity. In order to understand how this may occur, we have developed a new assay that can track parental histones through replication. Using this assay we will define whether histone deposition is influenced by the asymmetric nature of the replication fork and if there are specific mechanisms utilized by the cell to regulate where old and new histones are deposited. The final aspect of this proposal is to develop a new technology to map how individual replication forks proceed across genomes. Our understanding of DNA replication and its relationship with chromatin structure, nuclear organization and gene transcription has advanced significantly through the development of genome-wide assays. All genome-wide tools utilize large populations of cells meaning that they report on the average of the population; as such, many of the basic parameters that underlie how a replication fork progresses through chromatin are not known. We describe a new technique that captures and maps the positions and abundance of sister replication forks that initiated from the same replication origin. We show how this information can be used to generate an entirely new view of how replication proceeds across a genome.